title: 2-phenylethynesulfonamide Prevents Induction of Pro-inflammatory Factors and Attenuates LPS-induced Liver Injury by Targeting NHE1-Hsp70 Complex in Mice.
abstract: The endotoxin-mediated production of pro-inflammatory cytokines plays an important role in the pathogenesis of liver disorders. Heat shock protein (Hsp70) overexpression has established functions in lipopolysaccharide (LPS)-mediated inflammatory response. However, little is known about the role of Hsp70 activity in LPS signaling. We hypothesized that inhibition of Hsp70 substrate binding activity can ameliorate LPS-induced liver injury by decreasing induction of pro-inflammatory factors. In this study, C57/BL6 mice were injected intraperitoneally with LPS and 2-phenylethynesulfonamide (PES), an inhibitor of Hsp70 substrate binding activity. We found that i. PES prevented LPS-induced increase in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity, infiltration of inflammatory cells, and liver cell apoptosis; ii. PES reduced inducible nitric oxide synthase (iNOS) protein expression as well as serum nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) content in LPS-stimulated mice; iii. PES reduced the mRNA level of iNOS, TNF-α, and IL-6 in LPS-stimulated liver. iiii. PES attenuated the degradation of inhibitor of κB-α (IκB-α) as well as the phosphorylation and nuclear translocation of nuclear factor-κB (NF-κB) in LPS-stimulated liver. Similar changes in the protein expression of inflammatory markers, IκB-α degradation, and NF-κB phosphorylation and nuclear translocation were observed in RAW 264.7 cells. Further mechanistic studies revealed that PES remarkably reduced the elevation of [Ca(2+)]i and intracellular pH value (pHi) in LPS-stimulated RAW 264.7 cells. Furthermore, PES significantly reduced the increase in Na(+)/H(+) exchanger 1 (NHE1) association to Hsp70 in LPS-stimulated macrophages and liver, suggesting that NHE1-Hsp70 interaction is required for the involvement of NHE1 in the inflammation response. In conclusion, inhibition of Hsp70 substrate binding activity in vivo reduces the induction of pro-inflammatory factors and prevents LPS-induced liver injury likely by disrupting NHE1-Hsp70 interaction which consequently reduces the activation of IκB-α-NF-κB pathway in liver.
authors.affiliation: Department of Pharmacology, School of Medicine, Nantong University, Nantong, Jiangsu, People's Republic of China.
authors.forename: Chao
authors.initials: C
authors.lastname: Huang
authors.forename: Jia
authors.initials: J
authors.lastname: Wang
authors.forename: Zhuo
authors.initials: Z
authors.lastname: Chen
authors.forename: Yuzhe
authors.initials: Y
authors.lastname: Wang
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
mesh_terms: D000410:Alanine Transaminase / Q000097:blood; D000818:Animals; D001219:Aspartate Aminotransferases / Q000097:blood; D027682:Cation Transport Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D056486:Chemical and Drug Induced Liver Injury / Q000473:pathology* / Q000517:prevention & control / Q000662:veterinary; D018840:HSP70 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020672:I-kappa B Proteins / Q000378:metabolism; D015850:Interleukin-6 / Q000097:blood / Q000235:genetics / Q000378:metabolism; D008070:Lipopolysaccharides / Q000633:toxicity*; D008099:Liver / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009569:Nitric Oxide / Q000097:blood; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D000067996:RAW 264.7 Cells; D015398:Signal Transduction / Q000187:drug effects; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology* / Q000627:therapeutic use; D014409:Tumor Necrosis Factor-alpha / Q000097:blood / Q000235:genetics / Q000378:metabolism
chemical_list: C545747:2-phenylacetylenesulfonamide; D027682:Cation Transport Proteins; D018840:HSP70 Heat-Shock Proteins; D020672:I-kappa B Proteins; D015850:Interleukin-6; D008070:Lipopolysaccharides; C490997:Slc9a1 protein, mouse; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers; D013449:Sulfonamides; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D001219:Aspartate Aminotransferases; D000410:Alanine Transaminase